Comparison of excitatory currents activated by different transmitters on crustacean muscle. II. Glutamate-activated currents and comparison with acetylcholine currents present on the same muscle

The properties of glutamate-activated excitatory currents on the gm6 muscle from the foregut of the spiny lobsters Panulirus argus and interruptus and the crab Cancer borealis were examined using either noise analysis, analysis of synaptic current decays, or slow iontophoretic currents. The properties of acetylcholine currents activated in nonjunctional regions of the gm6 muscle were also examined. At 12 degrees C and -80 mV, the predominant time constant of power spectra from glutamate-activated current noise was approximately 7 ms and the elementary conductance was approximately 34 pS. At 12 degrees C and -80 mV, the predominant time constant of acetylcholine- activated channels was approximately 11 ms with a conductance of approximately 12 pS. Focally recorded glutamatergic extracellular synaptic currents on the gm6 muscle decayed with time constants of approximately 7-8 ms at 12 degrees C and -80 mV. The decay time constant was prolonged e-fold about every 225-mV hyperpolarization in membrane potential. The Q10 of the time constant of the synaptic current decay was approximately 2.6. The voltage dependence of the steady-state conductance increase activated by iontophoretic application of glutamate has the opposite direction of the steady-state conductance activated by cholinergic agonists when compared on the gm6 muscles. The glutamate-activated conductance increase is diminished with hyperpolarization. The properties of the marine crustacean glutamate channels are discussed in relation to glutamate channels in other organisms and to the acetylcholine channels found on the gm6 muscle and the gm1 muscle of the decapod foregut (Lingle and Auerbach, 1983).     

The single-channel basis for the slow kinetics of synaptic currents in vertebrate slow muscle fibers

The time course of synaptic currents is significantly longer in slow than in fast twitch muscle fibers. To examine the underlying basis for these slow synaptic currents, single-channel recordings were made from the synapses of slow muscle fibers. Our analysis indicates that low conductance acetylcholine receptor (AChR) channels predominate in innervated slow fibers. The high level of expression of low conductance channels is in contrast to fast twitch fibers, in which these channels are expressed in significant numbers only in embryonic or denervated muscle. Analysis of the distribution of open durations for the low conductance channel class suggests that the open time of this AChR class is the major determinant in shaping the slow time course of synaptic current decay. The predominant contribution of low conductance channel openings to synaptic currents of slow muscle fibers indicates a well-defined physiological role for this class of AChRs.     

Comparison of excitatory currents activated by different transmitters on crustacean muscle. I. Acetylcholine-activated channels

The properties of acetylcholine-activated excitatory currents on the gm1 muscle of three marine decapod crustaceans, the spiny lobsters Panulirus argus and interruptus, and the crab Cancer borealis, were examined using either noise analysis, analysis of synaptic current decays, or analysis of the voltage dependence of ionophoretically activated cholinergic conductance increases. The apparent mean channel open time (tau n) obtained from noise analysis at -80 mV and 12 degrees C was approximately 13 ms; tau n was prolonged e-fold for about every 100-mV hyperpolarization in membrane potential; tau n was prolonged e- fold for every 10 degrees C decrease in temperature. Gamma, the single- channel conductance, at 12 degrees C was approximately 18 pS and was not affected by voltage; gamma was increased approximately 2.5-fold for every 10 degrees C increase in temperature. Synaptic currents decayed with a single exponential time course, and at -80 mV and 12 degrees C, the time constant of decay of synaptic currents, tau ejc, was approximately 14-15 ms and was prolonged e-fold about every 140-mV hyperpolarization; tau ejc was prolonged about e-fold for every 10 degrees C decrease in temperature. The voltage dependence of the amplitude of steady-state cholinergic currents suggests that the total conductance increase produced by cholinergic agonists is increased with hyperpolarization. Compared with glutamate channels found on similar decapod muscles (see the following article), the acetylcholine channels stay open longer, conduct ions more slowly, and are more sensitive to changes in the membrane potential.     

Factors determining the duration of a single postsynaptic response in neuromuscular junctions

Parameters of single acetylcholine-activated ionic channels and the time course of miniature end-plate currents (MEPC) were compared in experiments on fast and slow lamprey, frog, chicken, and rat muscle fibers. The mean open time of the channels was shown to be the principal, but not the only factor determining the duration of MEPC. The role of the remaining factors and, in particular, of insufficiency of acetylcholinesterase activity, in slow muscle fibers and also in "giant" MEPC generation, is much greater than in fast fibers or during ordinary MEPC generation. Relatively low acetylcholinesterase activity favors asynchronous interaction between acetylcholine molecules and receptors, which delays the time course of synaptic responses. Mechanisms of acceleration of MEPC decay under the influence of -bungarotoxin and D-tubocurarine, and also the conditions for MEPC generation in different regions of the neuromuscular junction are discussed.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 16, No. 5, pp. 590–602, September–October, 1984.     

Endplate currents are shortened by octanol: Possible role of membrane lipid

The time constant of the decay phase of miniature endplate currents is shortened in the presence of octanol (mM). A change in the power spectrum of acetylcholine “noise” indicates that octanol shortens the duration of “elementary” conductance changes thought to depend on membrane conformational changes produced by acetylcholine. It is proposed that octanol causes these conformational changes to occur more rapidly by increasing the fluidity of membrane lipid.     

The frog interosseal muscle fiber as a new model for patch clamp studies of chemosensitive- and voltage-sensitive ion channels: actions of acetylcholine and batrachotoxin

The patch clamp technique was used to record the currents flowing through single ion channels in isolated frog muscle fibers. The majority of the acetylcholine (ACh)-activated channels had a conductance of 32 pS, although 20 pS channels were also occasionally observed. Lifetimes of ACh-activated channels increased with the transmembrane potential in the range from - 30 mV to - 105 mV. In these same fibers we also observed channels which were activated by low concentrations of batrachotoxin (BTX; 10 nM). These channels, presumed to be Na channels, had a conductance of 19 pS and opened at potentials at which Na channels would not normally open. A notable feature of these BTX-activated channels was that they opened and closed repeatedly. Therefore, it appears that the toxin, in addition to activating Na channels, also blocks the inactivation process. The physiological properties of these channels reveal significant differences between the ion channels of tissue-cultured and mature tissues.     

Numerical analysis of the voltage-clamp technique applied to frog neuromuscular junctions.

The nonlinear cable equation was solved numerically by means of an implicit procedure. The correlation between end-plate length and fiber diameter was determined in frog (Rana pipiens) sartorius muscles stained with gold chloride (Löwit, 1875). The diameter of the fibers stained by the Löwit method was 80 (74-85) micron (median and its 95% confidence interval for 52 fibers), the length of the end plates in the same fibers was 382 (353-417) micron. The fibers simulated were 80 micron in diameter. To solve the equation the muscle fibers were represented by 500 segments 20 micron long, and the equation was solved in steps of 10 microseconds; a double exponential function was incorporated to the first seven segments to represent the neuromuscular junction. The potential of the first segment of the cable was set to the clamping level and the membrane potential of the remaining segments calculated. The current needed to hold the first segment was estimated by adding the current flowing through the first segment to the current flowing from it to the second segment. Our results indicate that the lack of space clamp in the point voltage-clamp studies of the frog neuromuscular junction introduces serious errors in the estimates of the end-plate conductance value, the kinetics of the conductance changes, and the reversal potential of the end-plate currents. The possibility of an efficient voltage-clamp technique is also explored. Our calculations suggest that the study of end-plate current and conductance is possible with little error if the end-plate potential is controlled at both ends of the synaptic area simultaneously.     

Automated detection of elementary calcium release events using the á trous wavelet transform

We developed an algorithm for the automated detection and analysis of elementary Ca2+ release events (ECRE) based on the two-dimensional nondecimated wavelet transform. The transform is computed with the "à trous" algorithm using the cubic B-spline as the basis function and yields a multiresolution analysis of the image. This transform allows for highly efficient noise reduction while preserving signal amplitudes. ECRE detection is performed at the wavelet levels, thus using the whole spectral information contained in the image. The algorithm was tested on synthetic data at different noise levels as well as on experimental data of ECRE. The noise dependence of the statistical properties of the algorithm (detection sensitivity and reliability) was determined from synthetic data and detection parameters were selected to optimize the detection of experimental ECRE. The wavelet-based method shows considerably higher detection sensitivity and less false-positive counts than previously employed methods. It allows a more efficient detection of elementary Ca2+ release events than conventional methods, in particular in the presence of elevated background noise levels. The subsequent analysis of the morphological parameters of ECRE is reliably reproduced by the analysis procedure that is applied to the median filtered raw data. Testing the algorithm more rigorously showed that event parameter histograms (amplitude, rise time, full duration at half-maximum, and full width at half-maximum) were faithfully extracted from synthetic, "in-focus" and "out-of-focus" line scan sparks. Most importantly, ECRE obtained with laser scanning confocal microscopy of chemically skinned mammalian skeletal muscle fibers could be analyzed automatically to reproducibly establish event parameter histograms. In summary, our method provides a new valuable tool for highly reliable automated detection of ECRE in muscle but can also be adapted to other preparations.     

Gating of Na channels. Inactivation modifiers discriminate among models

Macroscopic Na currents were recorded from N18 neuroblastoma cells by the whole-cell voltage-clamp technique. Inactivation of the Na currents was removed by intracellular application of proteolytic enzymes, trypsin, alpha-chymotrypsin, papain, or ficin, or bath application of N-bromoacetamide. Unlike what has been reported in squid giant axons and frog skeletal muscle fibers, these treatments often increased Na currents at all test pulse potentials. In addition, removal of inactivation gating shifted the midpoint of the peak Na conductance-voltage curve in the negative direction by 26 mV on average and greatly prolonged the rising phase of Na currents for small depolarizations. Polypeptide toxins from Leiurus quinquestriatus scorpion and Goniopora coral, which slow inactivation in adult nerve and muscle cells, also increase the peak Na conductance and shift the peak conductance curve in the negative direction by 7-10 mV in neuroblastoma cells. Control experiments argue against ascribing the shifts to series resistance artifacts or to spontaneous changes of the voltage dependence of Na channel kinetics. The negative shift of the peak conductance curve, the increase of peak Na currents, and the prolongation of the rise at small depolarization after removal of inactivation are consistent with gating kinetic models for neuroblastoma cell Na channels, where inactivation follows nearly irreversible activation with a relatively high, voltage-independent rate constant and Na channels open only once in a depolarization. As the same kind of experiment does not give apparent shifting of activation and prolongation of the rising phase of Na currents in adult axon and muscle membranes, the Na channels of these other membranes probably open more than once in a depolarization.     

Electrophysiology of a chick neuronal nicotinic acetylcholine receptor expressed in Xenopus oocytes after cDNA injection

Brain nicotinic acetylcholine receptors (nAChRs) are made up of protein subunits that differ from those constituting muscle nAChRs. To characterize the physiological properties of one class of avian brain nicotinic receptor, we injected the nuclei of Xenopus oocytes with full-length cDNAs for the ligand binding (alpha 4) and structural (n alpha) subunits. Injected oocytes had large ACh-induced currents in the microampere range that were insensitive to alpha-bungarotoxin, as expected for neuronal nAChRs. We found that these brain nAChRs incorporate at least two alpha 4 subunits and that their functional properties differ from muscle nAChRs in at least two respects: the elementary conductance is considerably smaller (20 pS), and channels in outside out patches stop functioning within a few minutes.     

The kinetics of local anesthetic blockade of end-plate channels

The effect of the local anesthetic QX222 on the kinetics of miniature end-plate currents (MEPC)and acetylcholine-induced end-plate current fluctuations was studied in voltage-clamped frog cutaneous pectoris neuromuscular junctions. The rate constants for a kinetic scheme of local anesthetic blockage of end-plate channels were calculated from the MEPC decay parameters. At 18 degrees C the blocking rate constant was 1.1 +/- 0.3 x 10(7) exp (-0.009 +/- 0.003 x V)s -1M-1, and the unblocking rate constant was 5.7 +/- 0.6 exp (0.011 +/- 0.002 x V)s -1. The dissociation constant was close to 10 microM at -80 mV. End-plate fluctuations indicated that the local anesthetic QX222 lowered the effective single-channel conductance, suggesting a finite blocked state conductance that was calculated to be 1.6 pS. The apparent differences between QX222 interaction with end-plate and extrajunctional channels are discussed.     

Cluster organization and pore structure of ion channels formed by beticolin 3, a nonpeptidic fungal toxin

Beticolin 3 (B3) belongs to a family of nonpeptidic phytotoxins produced by the fungus Cercospora beticola, which present a broad spectrum of cytotoxic effects. We report here that, at cytotoxic concentration (10 microM), B3 formed voltage-independent, weakly selective ion channels with multiple conductance levels in planar lipid bilayers. In symmetrical standard solutions, conductance values of the first levels were, respectively, 16 +/- 1 pS, 32 +/- 2 pS, and 57 +/- 2 pS (n = 4) and so on, any conductance level being roughly twice the lower one. Whether a cluster organization of elementary channels or different channel structures underlies this particular property was addressed by investigating the ionic selectivity and the pore size corresponding to the first three conductance levels. Both selectivity and pore size were found to be almost independent of the conductance level. This indicated that multiple conductance behavior resulted from a cluster organization of "B3 elementary channels." According to the estimated pore size and analyses of x-ray diffraction of B3 microcrystals, a structural model for "B3 elementary channels" is proposed. The ability to form channels is likely to be involved in the biological activity of beticolins.     

Nicardipine inhibits axon conduction but causes dual changes of acetylcholine release in the mouse motor nerve

The effects of nicardipine, a dihydropyridine Ca2(+)-channel antagonist, on neuromuscular transmission and impulse-evoked release of acetylcholine were compared with those of nifedipine. In the isolated mouse phrenic nerve diaphragm, nicardipine (50 microM), but not nifedipine (100 microM), induced neuromuscular block, fade of tetanic contraction, and dropout or all-or-none block of end-plate potentials. Nicardipine had no significant effect on the resting membrane potential and the amplitude of miniature end-plate potentials but increased the frequency and caused the appearance of large size miniature potentials. The quantal contents of evoked end-plate potentials were increased. In the presence of tubocurarine, however, nicardipine depressed the amplitude of end-plate potentials. The compound nerve action potential was also decreased. It is concluded that nicardipine blocks neuromuscular transmission by acting on Na+ channels and inhibits axonal conduction. Nicardipine appeared to affect the evoked release of acetylcholine by dual mechanisms, i.e., an enhancement presumably by an agonist action on Ca2+ channels, like Bay K 8644 and nifedipine, and inhibition by an effect on Na+ channels, like verapamil and diltiazem. In contrast with its inactivity on the amplitude of miniature end-plate potentials, depolarization of the end plate in response to succinylcholine was greatly depressed. The contractile response of baby chick biventer cervicis muscle to exogenous acetylcholine was noncompetitively antagonized by nicardipine (10 microM), but was unaffected by nifedipine (30 microM). These results may implicate that nicardipine blocks the postsynaptic acetylcholine receptor channel by enhancing receptor desensitization or by a use-dependent effect.     

Increased excitability of acidified skeletal muscle: role of chloride conductance

Generation of the action potentials (AP) necessary to activate skeletal muscle fibers requires that inward membrane currents exceed outward currents and thereby depolarize the fibers to the voltage threshold for AP generation. Excitability therefore depends on both excitatory Na+ currents and inhibitory K+ and Cl- currents. During intensive exercise, active muscle loses K+ and extracellular K+ ([K+]o) increases. Since high [K+]o leads to depolarization and ensuing inactivation of voltage-gated Na+ channels and loss of excitability in isolated muscles, exercise-induced loss of K+ is likely to reduce muscle excitability and thereby contribute to muscle fatigue in vivo. Intensive exercise, however, also leads to muscle acidification, which recently was shown to recover excitability in isolated K(+)-depressed muscles of the rat. Here we show that in rat soleus muscles at 11 mM K+, the almost complete recovery of compound action potentials and force with muscle acidification (CO2 changed from 5 to 24%) was associated with reduced chloride conductance (1731 +/- 151 to 938 +/- 64 microS/cm2, P < 0.01) but not with changes in potassium conductance (405 +/- 20 to 455 +/- 30 microS/cm2, P < 0.16). Furthermore, acidification reduced the rheobase current by 26% at 4 mM K+ and increased the number of excitable fibers at elevated [K+]o. At 11 mM K+ and normal pH, a recovery of excitability and force similar to the observations with muscle acidification could be induced by reducing extracellular Cl- or by blocking the major muscle Cl- channel, ClC-1, with 30 microM 9-AC. It is concluded that recovery of excitability in K(+)-depressed muscles induced by muscle acidification is related to reduction in the inhibitory Cl- currents, possibly through inhibition of ClC-1 channels, and acidosis thereby reduces the Na+ current needed to generate and propagate an AP. Thus short term regulation of Cl- channels is important for maintenance of excitability in working muscle.     

Junctional and extrajunctional membrane channels activated by GABA in locust muscle fibres

Iontophoretic application of GABA to voltage-clamped locust muscle fibres has demonstrated the presence of both extrajunctional and junctional GABA receptors. Extrajunctional GABA receptors are distinct from extrajunctional glutamate receptors which also occur in these muscle fibres. Inward GABA currents are nonlinearly dependent on membrane potential. Analysis of membrane current noise produced by iontophoretic GABA application shows that for junctional and extrajunctional GABA receptors the mean channel lifetime is 3-4 ms and the single-channel conductance is approximately 22 pS at - 80 mV (T = 21 degrees C). The mean lifetime as previously demonstrated for glutamate-sensitive excitatory channels in locust muscle fibres.     

Chloride action potentials and currents in embryonic skeletal muscle of the chick

Chloride-dependent action potentials were elicited from embryonic skeletal muscle fibers of the chick during the last week of in ovo development. The duration of the action potentials was extremely long (greater than 8 sec). The action potentials were reversibly blocked by the stilbene derivative, SITS, a specific blocker of chloride permeability. Using patch clamp pipettes, in which the intracellular chloride concentration was controlled and with other types of ion channels blocked, the membrane potential at the peak of the action potential closely coincided with the chloride equilibrium potential calculated from the Nernst equation. These data indicate that activation of a chloride-selective conductance underlies the long duration action potential. The occurrence of the chloride-dependent action potential was found to increase during embryonic development. The percentage of fibers that displayed the action potential increased from approximately 20% at embryonic day 13 to approximately 70% at hatching. Chloride-dependent action potentials were not found in adult fibers. The voltage and time-dependent currents underlying the action potential were recorded under voltage clamp using the whole-cell version of the patch pipette technique. The reversal potential of the currents was found to shift with the chloride concentration gradient in a manner predicted by the Nernst equation, and the currents were blocked by SITS. These data indicate that chloride ions were the charge carriers. The conductance was activated by depolarization and exhibited very slow activation and deactivation kinetics.     

Computer simulation of the motoneuron pool–muscle complex. I. Input system and motoneuron pool

 The aim of the present study was to simulate the input system and the motoneuron (MN) pool of the MN pool–muscle complex (MNPMC). Input fibers, which can originate from command centers in the central nervous system or from sensory organs, activate the MN pool. They generate sequences of action potentials, the frequency of which is proportional to a time-dependent activation factor (which is an input to the model). Different connection patterns between the input fibers and motor units (MUs) are allowed. For simplicity and since no precise experimental data are available, 70 input fibers and 4 boutons per fiber and MN are simulated (this corresponds approximately to the monosynaptic group-Ia input of the cat medial gastrocnemius muscle). Each bouton generates the same conductance change in the postsynaptic membrane. The MNs are modeled with a single compartment and a homogenous membrane. According to experimental data, the membrane leakage conductance and capacitance are MU dependent. Since the precise relation is unknown: (a) the computed relation between MU contraction force and the MN leakage conductance was taken from a steady-state MNPMC model, and (b) the capacitance was assumed to be proportional to the leakage conductance. The MN membrane includes time- and voltage-dependent ionic channels (fast and slow K⁺ and low- and high-threshold Ca²⁺ channels). The density and time constant of the slow K⁺ channels and the density of the Ca²⁺ channels were fitted to approximate afterhyperpolarization characteristics and frequency-injected current relations of type-identified cat MNs. If the membrane reaches a voltage threshold the MNs generate action potentials, which were simulated by voltage pulses. The activation of the MN pool of the human first dorsal interosseus muscle was simulated with injected and synaptic currents in order to illustrate the size principle, synaptic noise, and other features of muscle activation. It is concluded that the present model reproduces the main properties of the input–output relations of different MN types within a muscle. Together with the simulation of the muscle force and the surface EMG, which will be published in subsequent papers, it will be a powerful tool for reproducing experiments on the motor system and investigating functional mechanisms of motor control. Received: 17 April 2001 / Accepted in revised form: 6 November 2001     

Effects of quantal content,membrane potential,and cholinoceptor density on the time course of end-plate currents in the rat under during acetylcholinesterase inhibition

The time-course of multiquantal end-plate currents (EPCs) was compared with monoquantal synaptic responses, i.e., miniature end-plate currents (MEPCs), in voltage-clamped rat diaphragm muscle fibers. In the presence of active acetylcholinesterase (AChE), the time constant of the decay of EPCs, that were composed of 25–140 quanta, was 1.2 times greater than that of MEPCs. After inhibition of AChE with armine or proserine the decay of the EPC was longer than the decay of the MEPC by 10–100 times, and unlike the MEPC, in the majority of synapses it could be described by the sum of two (n=34) or three (n=9) exponentials: monoexponential EPCs were noted in only three cases. The nature and duration of the EPC decay depended on its quantal content. After a reduction in the quantal content a three-exponential EPC decay could be successively reduced to a two- and a mono-exponential decay. A ,slow, component of the EPC decay, unlike the MEPC decay, was extremely sensitive to changes in the membrane potential, and extracellular magnesium ion concentration. When the cholinoceptors were irreversibly blocked by -bungarotoxin the MEPC decay accelerated, and the monoexponential EPC decay initially slowed down before accelerating, but even during a profound blockade the open-times of the ion channels were not affected. It therefore appears that during the generation of multiquantal EPCs when AChE is inhibited, not only does the synchronicity of the ion channel opening change, but so do their kinetics, possibly because of ion channel blockade by endogenous acetylcholine.S. V. Kurashov Institute of Medicine, Russian Ministry of Public Health, Kazan. Translated from Neirofiziologiya, Vol. 24, No. 3, pp. 269–279, May–June, 1992.     

Characteristics of postsynaptic potentials and ionic currents in synapses of fast and slow rat muscle fibers

Miniature end-plate currents and potentials (MEPPs and MEPCs, respectively) were recorded in fast and slow rat muscle fibers by extracellular focal recording and voltage clamp techniques. The rise time and the half-decay time of these potentials and currents were 1.3–1.4 times greater in slow fibers than in fast. A similar difference, but lesser in degree, also was observed after inhibition of acetylcholinesterase. Decline of the end-plate currents remained, generally speaking, exponential and its rate depended on the clamped voltage. The percentage distribution of fibers of different types by duration of MEPP and MEPC in fast and slow muscles correlated with the percentage distribution of fibers identified in these muscles on the basis of other parameters. Factors determining the time course of the responses (acetylcholinesterase activity, length of diffusion pathways, differences in passive electrical properties of the membrane), and their importance for synapses of different types, are discussed.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 12, No. 6, pp. 627–636, November–December, 1980.     

Open channel noise. I. Noise in acetylcholine receptor currents suggests conformational fluctuations.

The random passage of ions through an open channel is expected to result in shot noise fluctuations in the channel current. The patch-clamp technique now allows fluctuations of this size to be observed in single-channel currents. In the experiments reported here the acetylcholine-induced currents in cultured rat muscle cells were analyzed; fluctuations were found that were considerably larger than expected for shot noise. A low-frequency component, which was fitted with a Lorentzian, was examined in detail; it appears to arise from fluctuations in channel conductance of approximately 3% on a time scale of 1 ms. The characteristic relaxation time is voltage dependent and temperature dependent (Q10 approximately equal to 3) suggesting that the fluctuations arise from conformational fluctuations in the channel protein.